Fsh

ABSTRACT

Biologically active heterodimeric human FSH composed of an alpha subunit and a beta subunit, each subunit being synthesized by a cell having an expression vector containing heterologous DNA encoding the subunit.

BACKGROUND OF THE INVENTION

This application is a continuation-in-part of copending application Ser.No. 548,228 entitled "Heteropolymeric Protein" filed Nov. 2, 1983, nowpending.

This invention relates to the use of recombinant DNA techniques toproduce heteropolymeric proteins.

Various polypeptide chains have been expressed, via recombinant DNAtechnology, in host cells such as bacteria, yeast, and culturedmammalian cells. Fiddes, J. C. and Goodman, H. M. (1979) Nature Vol.281, pg. 351-356 and Fiddes, J. C. and Goodman, H. M. (1980) Nature Vol.286, pg. 684-687 describe the cloning of, respectively, the alpha andbeta subunits of human choriogonadotropin (hCG).

Kaname U.S. Pat. No. 4,383,036 describes a process for producing hCG inwhich human lymphoblastoid cells are implanted into a laboratory animal,harvested from the animal, and cultured in vitro; accumulated hCG isthen harvested from the culture.

SUMMARY OF THE INVENTION

In general the invention features the biologically active heterodimerichuman fertility hormone follicle stimulating hormone ("FSH") whichincludes an alpha subunit and a beta subunit, each subunit beingsynthesized by a cell having an expression vector containingheterologuous DNA encoding the subunit.

The term "expression vector" refers to a cloning vector which includesheterologous (to the vector) DNA under the control of sequences whichpermit expression in a host cell. Such vectors include replicatingviruses, plasmids, and phages. Preferred vectors are those containing atleast the 69% transforming region, and most preferably all, of thebovine papilloma virus genome.

The invention permits the production of biologically activeheterodimeric FSH from a single culture of transformed cells. Theproduction of both subunits of FSH in the same cell eliminates thenecessity of recombining subunits from separate cultures to assemble anactive heterodimeric molecule. The system also allows production of FSH,in a single culture, which undergoes, in the culture, post-translationalmodification, e.g. glycosylation and proteolytic processing, foractivity or stability.

In preferred embodiments, each expression vector is autonomouslyreplicating, i.e., not integrated into the chromosome of the host cell.The use of autonomously replicating expression vectors preventsundesirable influence of the desired coding regions by control sequencesin the host chromosome.

Other advantages and features of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

We turn now to the preferred embodiments of the invention, first brieflydescribing the drawings thereof.

DRAWINGS

FIG. 1 is a diagrammatic illustration of the construction of the plasmidpRF375.

FIG. 2 is a partial restriction map of the lambda clone 15B and the betaFSH-containing 6.8 kb EcoRI-BamHI fragment that is inserted into pBR322.

FIG. 3 is a partial restriction map of the beta FSH coding region andthe BamHI fragment that is inserted into a BPV based expression vector.

FIG. 4 is a diagrammatic illustration of the construction of theBPV-containing plasmid CL28FSH2.8BPV, encoding the beta subunit of FSH.

STRUCTURE

The cloning vectors of the invention have the general structure recitedin the Summary of the Invention, above. Preferred vectors have thestructures shown in the Figures, and are described in more detail below.

CONSTRUCTION OF CLONING VECTORS Isolation of cDNA Clones Encoding theCommon Alpha Subunit

In order to produce the heterodimeric FSH of the invention, the alphasubunit of human chorionic gonadotropin (hCG) first is isolated; thealpha subunit is common to the fertility hormones hCG, luteinizinghormone (LH), and FSH.

All of the techniques used herein are described in detail in Maniatis etal. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory), hereby incorporated by reference.

RNA is extracted from placental tissue by the following method.Homogenization of the tissue is carried out in a 1:1 mixture ofphenol:100 mM Na-acetate (pH 5.5) containing 1 mM EDTA, that has beenwarmed to 60° C. for 20 min. After cooling on ice for 10 min., thephases are separated by centrifugation. The hot phenol extraction isrepeated twice more followed by two extractions with chloroform.

RNA is precipitated from the final aqueous phase by the addition of 2.5volumes of ethanol.

In order to enrich for poly A+mRNA, placental RNA is passed over oligo(dT)-cellulose in 0.5M NaCl buffered with 10 mM Tris-HCl, pH 7.5, andwashed with the same solution. Poly A+mRNA is eluted with 10 mM Tris-HCl(pH 7.5), 1 mM EDTA, 0.05% SDS and precipitated twice with ethanol.Typical initial yields are 1.5-2.0 mg of total RNA per g of tissue, ofwhich about 2% is poly A+mRNA.

Placental cDNA libraries are constructed by reverse transcription ofplacental mRNA, second strand synthesis using E. coli DNA polymerase I(large fragment), treatment with SI nuclease, and homopolymer tailing(dC) with terminal deoxynucleotidyl transferase; all such procedures areby conventional techniques.

In a typical preparation, 20-30% conversion of mRNA to single strand(ss) cDNA; 70% resistance to digestion with nuclease S1 after secondstrand synthesis; and dC "tails" of ten to twenty-five bases in length,are obtained. These cDNA molecules are then annealed to DNA fragments ofthe plasmid pBR 322, which has been digested with PstI, and to which dG"tails" have been added. These recombinant plasmids are then used totransform E. coli cells to generate a cDNA library (transformed cellsare selected on the basis of tetracycline resistance).

In order to identify the human alpha hCG clone, a 219 bp fragment of amouse alpha thyroid stimulating hormone (TSH) clone is used as ahybridization probe. This probe has 77% sequence homology with the humanclone. It is radioactively labeled by nick translation and hybridized tothe cDNA library under conditions that take into account the extent ofhomology. Strongly hybridizing clones are analyzed by restrictionmapping and clones containing the complete coding sequence of alpha hCGare verified by DNA sequencing.

CONSTRUCTION OF PLASMID pRF375

Referring to FIG. 1, the plasmid CL28 (identical to plasmid JYMMT(E);Hamer et al. (1983) J. Mol. Applied Gen. 1, 273-288), containing themurine metallothionein promoter, SV40 DNA, and pBR322 sequences, is cutwith the restriction endonuclease BglII. At this site is inserted thecDNA clone of alpha hCG, containing untranslated regions of about 10 and220 b at its 5' and 3' ends, respectively. This clone has beengenetically engineered by the addition of synthetic BamHI linkers at itstermini.

The resulting plasmid pRF302 is digested with restriction enzymes BamHIand SalI to release the SV40 DNA sequence.

Plasmid pB2-2, which contains the entire BPV genome, and some pBR322sequences, is digested with BamHI and SalI to yield the BPV genome withBamHI/SalI ends; this fragment is ligated into pRF302 containing themetallothionein-hCG sequences.

Following transformation of E. coli, plasmid pRF375 is identified andisolated. It encodes the common alpha subunit under the control of themouse metallothionein promoter.

Isolation of the Human beta FSH Gene

A human genomic library in phage lambda (Lawn et al., 1978, Cell 15, p.1157-1174) is screened using "guessed" long probes. The idea behind suchprobes, set forth in Jaye et al. (1983) Nucleic Acids Research 11(8),2325, is that if the amino acid sequence of a desired protein is atleast partially known, a long probe can be constructed in which educatedguesses are made as to the triplet encoding any amino acid which can beencoded by more than one, and not more than four, different triplets.Any correct guesses increase the amount of homology, and improve thespecificity, of the results.

To isolate desired regions of DNA, two labeled 45-mer probes are used.TB36, homologous with amino acids 56-70 of human beta FSH; and TB21,homologous with amino acids 73-87. These probes have the followingnucleotide compositions (corresponding amino acids are also given):

    ______________________________________                                        TB36:        Val--Tyr--Glu--Thr--Val--Lys--Val--                              (AA56- 3'    CAC ATG CTC TGG CAC TCT CAC                                      70)                                                                                        Pro--Gly--Cys--Ala--His--His--Ala--Asp                                        GGT CCG ACG CGG GTG GTG CGA CTG 5'                               TB21:        Tyr--Thr--Tyr--Pro--Val--Ala--Thr--                              (AA73- 3'    ATG TGC ATG GGT CAC CGA TGT                                      87)                                                                                        Glu--Cys--His--Cys--Gly--Lys--Cys--Asp                                        CTC ACA GTG ACG CCG TTT ACG CTG 5'                               ______________________________________                                    

The above probes are used to screen the human genomic library asfollows. TB21 is labeled with ³² P and used to screen approximately5×10⁵ lambda plaques on duplicate filters by the in situ plaquehybridization technique of Benton and Davis (1977) Science 196, 180-182.The prehybridization solution is maintained at 55° C. for several hoursand has the following composition: 0.75M NaCl; 0.15M Tris/HCl, pH 8.0;10 mM EDTA; 5×Denhardt's Solution; 0.1% sodium pyrophosphate; 0.1% SDS;100 microgram/ml E. coli t-RNA. The hybridization solution has the samecomposition except that it is maintained overnight at 45° C., andcontains labeled probe in a concentration of about 0.5×10⁶ cpm/ml. Afterhybridization, the filters are washed four times in 1×SSC (=0.15M NaCl,0.015M Na₃ -citrate) and exposed to x-ray film.

This screening procedure yields 50 plaques which hybridize to TB21 onboth sets of filters. These 50 individual plaques are picked andcombined into 10 culture pools containing 5 plaques each. The 10cultures are grown and DNA is isolated from 50 ml phage lysates. ThisDNA is then digested with EcoRI and fractionated on two identical 1%agarose gels, after which it is transferred to nitrocellulose paperaccording to the method of Southern (1975) J. Mol. Biol. 98, 503-517.

The DNAs on the two filters are hybridized to ³² P labeled TB21 andTB36, respectively. Individual plaques from the pool containing arestriction fragment which strongly hybridizes to both probes are thenscreened according to the above procedure, except that the DNAs aredigested with EcoRI, BamHI, and EcoRI plus BamHI. In this way the 6.8 kbEcoRI-BamHI fragment containing human beta FSH is isolated.

A partial restriction map of clone 15B, containing the 6.8 kbEcoRI-BamHI fragment, is shown in FIG. 2. In order to locate theposition of the beta FSH sequences within the clone, the 6.8 kbEcoRI-BamHI fragment of clone 15B is subcloned into pBR322 to yieldplasmid p15B6.8R/B (FIG. 2). p15B6.8R/B is then digested with variousrestriction enzymes and the products are fractionated by agarose gelelectrophoresis using conventional methods. The DNA is blotted tonitrocellulose paper and hybridized to fragments of a porcine beta FSHcDNA clone labelled with ³² P by nick translation.

As shown in FIG. 2, the porcine beta FSH probe hybridizes to only twofragments of the human DNA, namely a 1.1 kb HindIII-KpnI and a 1.4 kbPstI fragment. Partial DNA sequencing of these two fragments shows thatthis DNA indeed codes for human beta FSH and that the entire codingregion for beta FSH is contained in these two fragments.

As shown by the restriction map of FIG. 3, the beta FSH coding sequenceis interrupted by an intervening sequence of approximately 1.6 kbbetween amino acids 35 and 36 of mature beta FSH. The nucleotidesequence of the entire human beta FSH coding region and some of theflanking and intervening sequences are given below. The amino acidsequence deduced from the nucleotide sequence is given for the codingregion. ##STR1##

Still referring to the above sequence, there is a box around the ATGinitiation codon of the 18 amino acid signal peptide, which is cleavedpost-translationally. The mature protein begins with the amino acid Asnencoded by the circled triplet AAT. The exon-intron boundaries aremarked by arrows; they are flanked by the concensus sequence GT for thesplice donor and AG for the splice acceptor site. There is a box aroundthe stop codon TAA, the end of the coding region.

Below is a reproduction of the above sequence not broken into triplets,showing restriction sites; the ATG beginning and the TAA ending thecoding region are boxed and the exon-intron junctions are marked byarrows. ##STR2##

Insertion of the Beta FSH DNA into a BPV-Based Expression Vector

Referring to FIG. 3, a synthetic BamHI linker is inserted at the DdeIsite of p15B6.8R/B, which is located 42 nucleotides 5' of the ATGinitiation codon. Referring to FIG. 4, p15B6.8R/B is digested with DdeIand treated with E. coli DNA polymerase (Klenow), after which it isligated to synthetic BamHI linkers and digested with BamHI. The 295 bpfragment containing the first exon of FSH is isolated and cloned intothe BamHI site of pBR322. The resulting plasmid pBR295Bam is digestedwith KpnI plus EcoRI plus AvaI and ligated to p15B6.8R/B which has beendigested with KpnI plus EcoRI plus SmaI. The ligation mix is then usedto transform E. coli, and the plasmid pBR2.8Bam containing the humanbeta FSH DNA sequence as a BamHI fragment is identified from among thetransformants by restriction mapping.

As shown in FIG. 4, expression plasmid CL28FSH2.8BPV is preparedaccording to the same method used to prepare pRF375 (FIG. 1), exceptthat the 2.8 kb BamHI fragment of pBR2.8Bam is used in place of thealpha hCG cDNA clone. Plasmid CL28FSH2.8BPV can be used to transformmammalian host cells using conventional methods, and human beta FSH canbe isolated and purified.

Transfection of Mouse Cells

To produce heterodimeric FSH using a mixed transfection, five ug of eachBPV plasmid, i.e., pRF375 (alpha subunit) and CL28FSH2.8BPV (beta FSH),are mixed and added to 0.5 ml of a 250 mM CaCl₂ solution containing 10ug of salmon sperm DNA as carrier. This mixture is bubbled into 0.5 mlof 280 mM NaCl, 50 mM Hepes and 1.5 mM sodium phosphate. The calciumphosphate precipitate is allowed to form for 30-40 minutes at roomtemperature.

24 hours prior to transfection, 5×10⁵ cells of mouse C127 cells(available from Dr. Dean Hamer, National Cancer Institute, NIH,Bethesda, MD) are placed in a 100 mm dish or T-75 flask. Immediatelybefore adding the exogenous DNA, the cells are fed with fresh medium(Dulbecco's Modified Medium, 10% fetal calf serum). One ml of calciumphosphate precipitate is added to each dish (10 ml), and the cells areincubated for 6-8 hours at 37° C.

The medium is aspirated and replaced with 5 ml of 20% glycerol inphosphate buffered saline, pH 7.0 (PBS) for 2 minutes at roomtemperature. The cells are washed with PBS, fed with 10 ml of medium,and incubated at 37° C. After 20-24 hours, the medium is changed andsubsequent refeeding of the cells is carried out every 3-4 days.Individual clones are grown in T-25 flasks. After 7-21 days, cell clonescan be transferred to larger flasks for analysis.

Deposits

The following, described above, has been deposited in the AgriculturalResearch Culture Collection (NRRL), Peoria, IL 61604: CL28FSH2.8BPV inE. coli, NRRL B-15923

The following, described above, has been deposited in the American TypeCulture Collection, Rockville, MD:

pRF375 in C127 cells, ATCC CRL 8401;

Applicants' assignee, Integrated Genetics, Inc., acknowledges itsresponsibility to replace these cultures should they die before the endof the term of a patent issued hereon, and its responsibility to notifythe ATCC and NRRL of the issuance of such a patent, at which time thedeposits will be made available to the public. Until that time thedeposits will be made available to the Commissioner of Patents under theterms of 37 CFR §1.14 and 35 USC §112.

Use

The transformed cell lines of the invention are used to produceglycosylated, biologically active heterodimeric human FSH, which ispurified from the cells and/or their culture media using conventionalpurification techniques. FSH has a number of well-known medical usesassociated with human fertility. For example, FSH can be used, alone orin conjunction with hCG or LH, to induce ovulation, or superovulationfor in vitro fertilization.

In addition, heterodimeric FSH, or the beta subunit alone, can be usedin diagnostic tests for fertility and pituitary functions.

FSH produced by recombinant cells has the advantage, compared to FSHobtained from natural sources, of being free from contamination by otherhuman proteins, in particular other fertility hormones.

Other embodiments are within the following claims. For example, ratherthan producing heterodimeric FSH by culturing cells containing twoseparate expression vectors, one encoding the alpha subunit and theother encoding the beta subunit, DNA encoding both subunits can beincluded in the same expression vector.

We claim:
 1. A method for producing biologically active, heterodimeric human FSH comprising culturing mammalian cells capable of glycosylating proteins, said cells comprising an expression vector encoding the alpha and beta subunits of said FSH.
 2. A vector comprising a DNA sequence encoding the beta subunit of human FSH. 